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Quantitative approaches to studying NK cell functional heterogeneity
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0002-6019-8157
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

It is commonly stated that the cell is the smallest functional unit of life. By analogy, then, the immune cell is the smallest functional unit of the immune system. Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. Many phenotypically distinct subpopulations of NK cells have been discovered, usually by dividing cells on the basis of cell-surface markers. These subpopulations are typically described as related to activation or developmental status of the cells. However, how these distinct phenotypes correlate with behavior in e.g. NK–target interactions is less widely understood. There is therefore a need to study NK cell behavior down at the single-cell level. The aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells.

Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they can be imaged over extended periods of time. We have performed experiments on both resting and IL-2-activated NK cells and quantified their cytotoxic behavior. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, which we termed “serial killers”, displayed faster and more effective cytotoxicity.

Also, we identified differences between resting and activated NK cells in regard to their migration and contact dynamics. Activated NK cells were found to more readily adhere to targets cells than did NK cells freshly isolated from peripheral blood. Apart form migration and contact dynamics, we have also quantified killing behavior, where NK cells can be seen to exhibit a behavior we term multiple lytic hits on the basis of analyzing target cell fluorescence profiles.

We have quantified these heterogeneities and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These methods, and automated single-cell analysis methods, will likely play a more important role in the study of immune responses in the future.

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , xiii, 35 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:21
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:kth:diva-146281ISBN: 978-91-7595-166-9 (print)OAI: oai:DiVA.org:kth-146281DiVA: diva2:723534
Presentation
2014-05-27, sal Air, Gamma, Science for Life Laboratory, Science for Life Laboratory, Solna, 13:00 (English)
Opponent
Supervisors
Note

QC 20140611

Available from: 2014-06-11 Created: 2014-06-11 Last updated: 2014-06-11Bibliographically approved
List of papers
1. Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
Open this publication in new window or tab >>Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
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2012 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, no OCT, 300- p.Article in journal (Refereed) Published
Abstract [en]

Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells.This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events.The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the population level.

Keyword
Cell migration, Cytotoxicity, Live cell imaging, Microchip, NK cell, Single cell, Ultrasound
National Category
Other Physics Topics
Identifiers
urn:nbn:se:kth:diva-119183 (URN)10.3389/fimmu.2012.00300 (DOI)2-s2.0-84874217873 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130311

Available from: 2013-03-11 Created: 2013-03-08 Last updated: 2017-12-06Bibliographically approved
2. Classification of human natural killer cells based on migration behavior and cytotoxic response
Open this publication in new window or tab >>Classification of human natural killer cells based on migration behavior and cytotoxic response
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2013 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 121, no 8, 1326-1334 p.Article in journal (Refereed) Published
Abstract [en]

Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

Keyword
Apoptosis, Cell Communication, Cell Degranulation, Cell Movement, HEK293 Cells, Humans, Immunophenotyping, Killer Cells, Natural, Lymphocyte Activation, Microchip Analytical Procedures, Models, Biological, Necrosis, T-Lymphocytes, Cytotoxic
National Category
Hematology
Identifiers
urn:nbn:se:kth:diva-125773 (URN)10.1182/blood-2012-06-439851 (DOI)000321750000017 ()2-s2.0-84874447340 (Scopus ID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130814

Available from: 2013-08-14 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
3. Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.
Open this publication in new window or tab >>Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.
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2014 (English)In: Frontiers in immunology, ISSN 1664-3224, Vol. 5, 80- p.Article in journal (Refereed) Published
Abstract [en]

Natural killer (NK) cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor-ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e., the immune synapse. We have studied the single-cell migration behavior and target-cell contact dynamics of resting and interleukin (IL)-2-activated human peripheral blood NK cells. Small populations of NK cells and target cells were confined in microwells and imaged by fluorescence microscopy for >8 h. Only the IL-2-activated population of NK cells showed efficient cytotoxicity against the human embryonic kidney 293T target cells. We found that although the average migration speeds were comparable, activated NK cells showed significantly more dynamic migration behavior, with more frequent transitions between periods of low and high motility. Resting NK cells formed fewer and weaker contacts with target cells, which manifested as shorter conjugation times and in many cases a complete lack of post-conjugation attachment to target cells. Activated NK cells were approximately twice as big as the resting cells, displayed a more migratory phenotype, and were more likely to employ "motile scanning" of the target-cell surface during conjugation. Taken together, our experiments quantify, at the single-cell level, how activation by IL-2 leads to altered NK cell cytotoxicity, migration behavior, and contact dynamics.

National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-146251 (URN)10.3389/fimmu.2014.00080 (DOI)000354057200001 ()24639676 (PubMedID)2-s2.0-84897939167 (Scopus ID)
Note

QC 20140611

Available from: 2014-06-11 Created: 2014-06-10 Last updated: 2017-01-09Bibliographically approved

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